Mycobacterium tuberculosis (M. TB). It is transmitted through respiratory droplets. Increased cholesterol level is a predisposing factor for TB. M. TB uses cholesterol in the host macrophage membranes to bind and enter the macrophages. Statins are the drugs that are prescribed to hyperlipidemic patients to maintain their lipid levels in the normal range, thereby reducing the risk of stroke and cardiovascular events. Moreover, statins aid in reducing the levels of cholesterol in human macrophages. Therefore, a reduction in the membrane cholesterol minimizes the entry of TB pathogen inside macrophages. Furthermore, acting as vitamin D3 analogs and positively influencing pancreatic beta-cell function in a chronic diabetic state, statins minimize the occurrence of M. TB infection among diabetic population as well. This review aims to provide a comprehensive detail of all in vitro, in vivo, and retrospective studies that investigated the effects of statins in relation to the prevention or treatment of TB infection.Tuberculosis (TB) is a chronic infection caused by  Mycobacterium tuberculosis (M. TB). In 2018, it was estimated that nearly 10 million people got infected with TB. It is one of the top 10 causes of death worldwide, even surpassing human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS). A total of 1.5 million deaths were reported from TB in 2018. Pakistan was among the eight countries that were responsible for two-thirds of the TB cases reported worldwide. Globally, the rate of TB infection is falling by 2% annually, and it has been estimated that nearly 58 million lives have been saved between 2000 to 2018 via adequate diagnosis and treatment of TB [Tuberculosis (TB) is a chronic infection caused by nt of TB . Conditint of TB ,5.et al. have explained the mechanism of action of statins in detail [Statins are the drugs that are prescribed to hyperlipidemic patients to maintain lipids levels in the normal range, thereby reducing the risk of stroke and cardiovascular events. They work by blocking 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate-limiting enzyme in the cholesterol synthesis pathway -6. In hiIncreased cholesterol levels is a predisposing factor for TB. M. TB uses cholesterol in the host macrophage membrane to bind and enter the macrophage -8. M. TBNo randomized controlled trial (RCT) has been carried out to study risk versus benefit of statins for TB. However, several new in vitro and in vivo studies have been published in the last few years. This review aims to provide comprehensive detail of all in vitro, in vivo, and retrospective studies that reported the effects of statins in relation to the prevention or treatment of TB infection.Why mycobacterium has a longer life-span inside macrophage?The cycle of TB infection begins with the dispersion of M. TB aerosols. There is a more likely risk of transmission when approximately one to 10 bacilli are dispersed through the air. M. TB, being a potent facultative intracellular pathogen, eludes host immunity and is taken up by the alveolar macrophages via phagocytosis . PhagocyWhat is the role of cholesterol in the development of TB infection?Cholesterol in the membrane of host macrophage has been regarded as a high-affinity docking site responsible for stable anchoring of M. TB pathogen prior to phagocytosis . M. TB eThe formation of a phagosome following the successful entry of M. TB pathogen inside macrophage is influenced by the amount of cholesterol in the plasma membrane. The premature formation of PI3P and its binding with Rab5 and other binding proteins occurs in a cholesterol dependant manner. The persistent nature of M. TB relies on its ability to inhibit phagosomal maturation. It resides inside a pathogen-friendly phagosome escaping lysosomal fusion, an event critical for bactericidal mechanisms. Following the entry via cholesterol-rich domains, mycobacteria are sequestered in the phagosomes . FurtherThe engagement of NADPH oxidase subunits with the phagosome is believed to be cholesterol-dependent . Hence, A sterol metabolism, utilizing host cholesterol, has been observed as a crucial step towards the development of M. TB infection -27. ThusHow does statin therapy prevent TB infection?Statins (HMG-CoA reductase inhibitors) aid in reducing the levels of cholesterol in human macrophages by various mechanisms . Some ofWhat do studies suggest regarding anti-TB effects of statin therapy?In Vitro Studieset al., where it was observed that fluvastatin modulates the release of type 1 T helper (Th1) and type 2 T helper (Th2) cytokines and consequently activates caspase-1 or leads to the secretion of interleukin (IL)-1β, IL-18 and interferon gamma (IFNγ) [et al. reported that lovastatin and fluvastatin interfere with the formation of lipid rafts by inhibiting tyrosine phosphorylation and expression of monosialotetrahexosylganglioside (GM1) and cluster of differentiation 69 (CD69) in gamma delta (γδ) T cells. This leads to inhibition of γδ T cells induced by M. TB antigens [The first study to highlight the potential effects of statins on infection by M. TB was conducted 20 years ago by Montero a (IFNγ) . The pera (IFNγ) . In 2009antigens . Howeverantigens -39.et al. studied the host protective mechanisms in individuals with hypercholesterolemia [Parihar erolemia . The stuerolemia .et al., in 2014, investigated the efficacy of atorvastatin (0.2-2µM) and simvastatin (0.2-2µM) with rifampicin (RIF) (1µg/mL) on the intracellular viability of mycobacteria (M. leprae and M. TB) within THP-1 macrophages [M. leprae strain, only atorvastatin showed an additional effect with RIF. The mechanism involved in the inhibition of mycobacterial growth was determined by testing the effect of atorvastatin on THP-1 macrophages infected by M. leprae. The results concluded that bactericidal effects in the cells infected by M. leprae were secondary to phagosomal arrest [Lobato rophages . After 7l arrest .et al. performed an experiment on J774 macrophages [To evaluate the tuberculocidal effect of simvastatin alone or in combination with first-line anti-TB drugs, Skerry rophages . After 2rophages . et al. studied the adjuvant effect of simvastatin with INH, RIF, and PZA on the duration of corrective treatment [In 2016, Dutta reatment . To detereatment .et al. analyzed the effects of simvastatin on the treatment of M. TB infection [Guerra-De-Blas nfection . Direct nfection .et al. conducted another study in 2019 [To determine whether statins can enhance the activity of anti-TB drugs against intracellular bacilli in macrophages, Dutta  in 2019 . They pe in 2019 . All in In Vivo StudiesAll in vivo studies depicting the effects of statins on infection by M. TB have been performed on mice. The first study to investigate M. TB infection in a statin-treated experimental model was performed on C57BL/6 mice (8-12 weeks of age) [et al. evaluated the ability of atorvastatin to potentiate the anti-bacterial effects of RIF [4 live M. leprae strains into plantar pads of each shepherd model of BALB/c mice. The mice were divided into six groups after one month of infection and were treated for five months. The control groups were not treated. Two other groups were treated with 40 mg/kg and 80 mg/kg of atorvastatin respectively. The other three groups received RIF (10 mg/kg and 1 mg/kg by gavage weekly) alone or a combination of RIF (1 mg/kg) and atorvastatin (80 mg/kg) daily. After six months, it was noticed that atorvastatin reduced bacterial replication and synergized anti-bacterial effects of RIF. Furthermore, no hepatotoxicity or muscle damage was observed with treatment [In 2014, Lobato s of RIF . They inet al. investigated whether the addition of simvastatin to the first-line anti-TB regimen shortens the duration of curative TB treatment [10 CFU). After the progression of infection for about six weeks, the mice were inoculated with RIF (10 mg/kg), INH (10 mg/kg), PZA (25 mg/kg) with and without the addition of simvastatin (25 mg/kg) for five days per week for eight weeks. After four and eight weeks of treatment, a significant reduction in bacterial load was noticed in the mice treated with a combination of simvastatin and standard anti-TB regimen. The number of lung colony-forming unit (CFU) reduced by 1 log10 on day 28 and 1.25 log10 on day 56. The results suggested that simvastatin can produce complementary anti-bacterial effects when combined with the first-line anti-TB regimen [Subsequently, Dutta reatment . BALB/c  regimen .et al. conducted another experiment on female C3HeB/FeJ mice, five to six weeks of age to determine the anti-TB activity of statins in mice [2 CFU/mouse lung. Six weeks after infection, mice were treated with INH (10 mg/kg), RIF (10 mg/kg), PZA (150 mg/kg) and ethambutol (EMB) (100 mg/kg) supplemented with simvastatin (90 mg/kg), fluvastatin (15 mg/kg) and pravastatin (50 and 90 mg/kg). The treatment was administered once daily for five days a week for eight weeks. Satisfactory results were observed as the adjunctive therapy with statins significantly reduced bacillary burdens in the lungs of mice. The number of lung CFU decreased to 1.28 log10, 1.16 log10, 0.78 log10, and 0.90 log10 by adjunctive treatment with simvastatin (90 mg/kg), pravastatin (90 mg/kg), pravastatin (50 mg/kg) and fluvastatin (15 mg/kg), respectively [In 2019, Dutta  in mice . The micectively . All in Retrospective StudiesSeveral retrospective cohorts and population-based case-control studies have highlighted the effects of statin on the risk of developing TB. Kang et al. , in 2014Lee et al.  conducteUsing the database of the Taiwan National Health Insurance Program, Lai et al.  conducteAnother population-based case-control study  conducteSu et al.  conducteet al. to investigate the effects of statins on the risk of developing TB and pneumonia in asthma-chronic pulmonary disease overlap syndrome (ACOS) patients from 2000 to 2010 [Subsequently, another retrospective cohort study was performed by Yeh  to 2010 . Cox pro to 2010 .et al. conducted a retrospective population-based cohort study in patients newly diagnosed with pulmonary TB [To examine the association between the use of lipid-lowering agents (LLAs) and outcomes of patients with pulmonary TB receiving anti-TB treatment, Chen onary TB . Using tonary TB .The most recently published retrospective population-based study assessed the effects of statins and non-statin LLAs on the risk of TB and herpes zoster among patients with type 2 DM . The parAll studies demonstrating the effects of statins on the risk of developing TB have been conducted in Korea and Taiwan. Hence, verification is required for generalizing results to other populations. In addition, the studies conducted to date are retrospective and contain information from databases, no updated statistics are available to support the findings and a causal relationship cannot be substantiated.In this review, we have gathered all in vivo, in vitro, and retrospective studies highlighting the tuberculocidal effects of statins and their adjunctive effects with first-line anti-TB therapy for the definitive cure of M. TB infection. Studies conducted on in vitro models have shown the immunomodulatory effects of statins and the dominant role of macrophages in resisting M. TB infection in the presence of statins. Moreover, it has been seen that statin therapy promotes phagolysosome maturation as well as aids autophagy. In addition, it has many anti-inflammatory effects, but the knowledge regarding its microbiological effects on the immune system needs to be researched. Studies in mice have shown that statin therapy can decrease the bacillary burden and shorten the duration of anti-TB treatment. However, no information on mechanisms through which statins enhance the anti-microbial effects of these drugs has been provided. We can hypothesize that statins might weaken mycobacterial cell wall making it more vulnerable to the standard TB regimen. In addition, statins may also bolster the immune response of the host and assist in the early eradication of the disease. Many retrospective studies have highlighted the effects of statin therapy on the risk of contracting TB infection. Albeit, these bactericidal effects are less potent than the standard anti-TB therapy. Chronic dose-dependent use of statins (>90 days) has been observed to be associated with further reduction in risk. Since all these researches have been conducted in Taiwan and Korea, the results cannot be generalized to the other populations due to lack of verification. Hence, prospective studies demonstrating the protective effects of statins on active TB should be conducted to identify the causal relationship.


Rhodnius prolixus as an experimental model, we have studied the involvement of insulin-like peptides (ILPs) and adipokinetic hormone (AKH) signaling in carbohydrate metabolism, focusing on the regulation of the trehalose-specific facilitated transporter (Rhopr-TRET), particularly in the ovaries. We find that trehalose stores in ovaries increase after feeding, synchronously with the beginning of vitellogenesis, but that the transcript expression of enzymes involved in trehalose synthesis show no changes between unfed and blood-fed animals. However, an eightfold increase in Rhopr-TRET transcript expression is observed in the ovaries post-blood meal. In vivo and ex vivo assays using exogenous insulins and Rhopr-AKH, reveal that Rhopr-TRET is up-regulated in ovaries by both peptide families. In accordance with these results, when ILP and AKH signaling cascades are impaired using RNA interference, Rhopr-TRET transcript is down-regulated. In addition, trehalose injection induces an up-regulation of Rhopr-TRET transcript expression and suggests an activation of insulin signaling. Overall, the results support the hypothesis of a direct trehalose uptake by ovaries from the hemolymph through Rhopr-TRET, regulated by ILP and/or AKH. We also show that Rhopr-TRET may work cooperatively with AKH signaling to support the release of trehalose from the ovaries into the hemolymph during the unfed (starved) condition. In conclusion, the results indicate that in females of R. prolixus, trehalose metabolism and its hormonal regulation by ILP and AKH play critical roles in adapting to different nutritional conditions and physiological states.Using the blood-sucking kissing bug,  Carbohydrates represent one of the main energy reserves in animal cells, which is why they participate as essential factors in various biological processes of all multicellular organisms . In inseIn insects, carbohydrate homeostasis is under strict hormonal regulation  led mainBlood-sucking insects usually need to process a large amount of blood in a short time to provide energy to support the optimal progress of physiological events related to growth, development, and reproduction. They also often have to overcome long periods of starvation between successful gorging . Insect Trypanosoma cruzi, the causal agent of Chagas disease. This illness is endemic in Latin American countries, but with globalization, this disease has spread throughout the world, and according to the World Health Organization, affects 6-7 million people worldwide  condition depending on availability of a blood-donating host.Triatomines (Hemiptera: Reduviidae) are a subfamily of blood-sucking insects with relevance in public health since they are vectors of orldwide . Furtherorldwide . In thisR. prolixus, AKH-signaling plays a critical role in modulating lipid levels (AKHR) transcript and ILP/IGF signaling activation in ovaries of R. prolixus has been reported  compared to those found for other nutrients, such as lipids and proteins (R. prolixus TRET (Rhopr-TRET), regulated by insulin and/or AKH-signaling, is important for the storage of carbohydrates by the ovaries. Also, we demonstrate that Rhopr-TRET could work cooperatively with the R. prolixus AKH (Rhopr-AKH) signaling pathway to promote the release of trehalose from the fat body and ovaries to the hemolymph during starvation. Overall, these results show that in R. prolixus, carbohydrate metabolism and its hormonal regulation may play critical roles in adapting to different physiological conditions, including reproduction and starvation.In d levels , and thed levels . The prereported , suggest sources . Althougproteins , the useR. prolixus taken from an established colony at the University of Toronto Mississauga. Insects were reared in incubators at 25°C under high humidity (∼50%). Standardized conditions of insect rearing were previously described  and then homogenized in 1.5 ml microtubes containing 200 μl of cold phosphate-buffered saline . The homogenates were centrifuged at 2,500 × g for 5 min at 4°C and pellets containing tissue debris were discarded. The resulting material was re-centrifuged at 12,000 × g for 10 min at 4°C and the supernatants collected for trehalose quantification. After immobilizing the insects on a slide using masking tape, 10 μL of hemolymph was collected using a Hamilton syringe  from cut ends of the legs while pressing gently on the abdomen. The hemolymph was placed in ice-cold microtubes and then diluted in cold anticoagulant solution  at a ratio of 1:5 (anticoagulant: hemolymph)  plus 20 μl of 1 M acetic acid were added, mixed and then centrifuged at 12,000 × g for 10 min at room temperature. The supernatants obtained were used to measure trehalose levels. One-hundred μl of each supernatant was incubated overnight at 37°C with 1 μl porcine kidney trehalase  to catalyze the conversion of trehalose into glucose, or with R. prolixus saline to determine the endogenous glucose content of the samples. Glucose contained in all the samples was determined using the glucose assay kit  according to the manufacturer’s protocol (30 μl of sample was added to 100 μl of glucose reagent solution). A standard curve using a 0–0.6 μg range of glucose was run in parallel with the experimental samples. The samples were quantified at 540 nm using a plate reader spectrophotometer . The trehalose concentration was then corrected for the amount of glucose present in the samples /2). The final trehalose concentration in each tissue or hemolymph sample was obtained by taking into consideration all dilutions used throughout the protocol. The results are shown as the mean ± standard error of the mean (SEM) .Adult female insects were dissected 10, 20, and 30 days post-ecdysis . One group of adult insects were fed and the tissues collected at 1, 2, 3, 4, and 5 days post-blood meal (representing the fed condition). Ovaries and ventral and dorsal fat bodies were dissected under cold molymph) . SamplesNilaparvata lugens (XP_022183984.1) and Cimex lectularius (XP_024085685.1), we obtained the putative Rhopr-TRET. To successfully amplify and confirm the 5′ and 3′ regions of Rhopr-TRET, 5′ and 3′ rapid amplification of cDNA ends (RACE) assays were performed using the SMARTer RACE 5′/3′ Kit . Briefly, fat bodies from fed females were dissected and placed in cold autoclaved phosphate buffered saline . Total RNA extraction was performed with Trizol reagent  according to the manufacturer’s recommendations. The final concentration and A260/280 ratio of purified RNA was measured using a DS-11 + spectrophotometer . All samples had a ratio between 1.9 and 2.0. RNA integrity, including potential degradation products and DNA contamination, was evaluated by electrophoresis in a 1% agarose gel . RNA was considered intact when the 18S rRNA band was observed. 5′- and 3′-RACE-ready cDNA were synthesized using the SMARTScribe Reverse Transcriptase, provided in the kit. Three specific pairs of primers were used in order to ensure specificity of the amplified sequence. The manufacturer-supplied protocol was followed to perform RACE reactions. The final products of the RACE reactions were later subjected to 2% agarose gel electrophoresis and the single products obtained of the expected sizes were sent for Sanger sequencing by Macrogen USA . The gene-specific primers utilized are shown in By BLAST  algorithm, the gene annotation from the RproC1.3 gene set  and usinRhopr-TRET, the predicted structural and biochemical features of the transporter were analyzed using online tools. To predict the exon–intron boundaries within the sequence, nucleotide BLAST (BLASTn) was performed using the R. prolixus genome deposited in VectorBase. Exon–Intron Graphic Maker was the tool used to make the exon map of Rhopr-TRET with the ORF1. The deduced amino acid sequence and molecular mass prediction were assessed using tools available on ExPASy2    . The tracations6 . In orde server7 . In addi server7 , potentirojects) , b, was 2 server . The str2 server .8 and later imported into the BOXSHADE 3.21 server9. Thirty-six amino acid sequences , Rhopr-TRET and 2 trehalases, membrane-bound (m-Tre) and soluble (s-Tre) enzymes , were assessed in the fat body and ovaries of females throughout different time points of the unfed and fed states. In addition, the Rhopr-AKH precursor (VectorBase: RPRC000416) and its receptor, Rhopr-AKHR (GenBank: KF534791), were analyzed throughout different time points of the unfed condition in fat body, ovaries and central nervous system . The spatial distribution of the Rhopr-TRET transcript in different tissues  was evaluated during the unfed condition (baseline stage). Tissue dissection was performed as previously reported . The conditions of the thermal cycler were: 10 min at 25°C, 120 min at 37°C, and 5 min at 85°C. qPCR assays were performed using an advanced master mix with super green low rox reagent , according to the manufacturer’s recommendations, using 4 pmol of sense and antisense primers in a final volume of 10 μl. The qPCR temperature-cycling profile was: initial denaturation 3 min at 95°C, followed by 39 cycles of 30 s at 94°C, 30 s at 58–60°C (depending on the pair of primers used), and 1 min at 72°C, followed by a final extension at 72°C for 10 min. Each reaction contained 3 technical replicates as well as a no template control and a no reverse transcriptase control. The reactions were carried out using a CFX384 Touch Real-Time PCR Detection System . Quantitative validation was analyzed by the 2–ΔΔCt method (r2) ranging from 0.8 to 1, and the dissociation curves always showed a single peak, indicating that a single cDNA product was amplified and excluding the possibility or tendency of the primers to form dimers. Specific target amplification was confirmed by automated DNA sequencing, as described above. The results are shown as the mean ± SEM .mRNA levels of reported , b. Briet method . 18S ribt method  and the ex vivo assays, ovaries of unfed females at 10 days post-ecdysis were incubated with human insulin , porcine insulin , R. prolixus ILP1  or with of the mammalian insulin receptor-specific activator bpV (phen) . Insulin stock solutions were solubilized in pH 3 with HCl and bpV (phen) in dimethyl sulfoxide (DMSO), according to the manufacturer protocol. To make working solutions, all the drugs were diluted in R. prolixus saline. The incubations were performed for 3 h or 12 h, as indicated, in 200 ml of Grace’s Insect Medium , at 30°C in the dark, as previously described . Stock solutions of Rhopr-AKH were dissolved in 50% 1-Methyl-2-Pyrrolidinone  and then stored at −20°C .For escribed . The finreported . In paraat −20°C . Rhopr-Aeriments . After iin vivo assays, unfed insects (10 days post-ecdysis) were injected into the hemocoel with 5 μl of 0.1 μg/μl porcine insulin  we previously demonstrated that unfed females of R. prolixus are in a sensitized state to respond to an increase of ILP levels by rapidly activating ILP signaling .For  insulin  or 0.01  insulin . As contignaling , and (b)ignaling  and, thuR. prolixus genome is deposited. Alignments with the best-matching sequences are shown and scored in Rhopr-IR1 (Vectorbase: RPRC006251) and Rhopr-AKHR (GenBank: KF534791), respectively, was previously demonstrated  were separated and injected (1 day after dsRNA treatment) into the hemocoel with 5 μl of 0.1 μg/μl porcine insulin, 0.01 μg/μl Rhopr-AKH or R. prolixus saline (control). Ovaries were removed under autoclaved PBS at 3 h after hormone injection and processed to RNA extraction and subsequent RT-qPCR to assess Rhopr-IR1, Rhopr-AKHR and Rhopr-TRET mRNA levels, as indicated. In all cases, the results are shown as the mean ± SEM . In addition, the percent survival of dsRhopr-AKHR injected insects during the unfed condition was analyzed (n = 15).To evaluate the potential regulation of insulin signaling on ctivated . In brieR. prolixus saline, or 5 μl of R. prolixus saline (control). Insects were dissected 3 h post-injection and the expression of Rhopr-TRET and of the peptides Rhopr-ILP1 (GenBank: KT896507.1), Rhopr-IGF  and Rhopr-AKH were measured in the ovaries, fat bodies or CNS, as indicated, by RT-qPCR. In addition, groups of dsRhopr-IR1 and dsARG treated insects were injected (1 day after dsRNA treatment) into the hemocoel with 5 μl of 70 μg/μl trehalose solution or 5 μl of R. prolixus saline (control). Fat bodies were removed under autoclaved PBS at 3 h post-injection and processed for RNA extraction and subsequent RT-qPCR to assess Rhopr-IR1 and Rhopr-TRET mRNA levels. In all cases, the results are shown as the mean ± SEM .In order to evaluate the effect of an increase in circulating trehalose on insulin and AKH signaling, unfed females (10 days post-ecdysis) were injected into the hemocoel with 5 μl of 70 μg/μl trehalose solution  diluted in 10). Before performing statistical analysis, we evaluated the data for normality and homogeneity of variance using the Shapiro–Wilk test, which showed that no transformations were needed, all datasets passed normality and homoscedasticity tests. Multiple group analysis was conducted by one-way ANOVA and Tukey’s test as post hoc test, and statistically significant difference between two groups were inferred using T-test. Differences in the survival curve were analyzed using the Log-Rank test. In all the cases, a p-value < 0.05 was considered statistically significant.All graphs were created using GraphPad Prism 9 . Our results by RACE show an open reading frame (ORF) to Rhopr-TRET of 1,461 bp, which encodes a 51.58-kDa protein of 477 amino acids  (Rhopr-TRET (RPRC007957) having the highest fold changes between fed and unfed insects .Recently, by transcriptome analysis we showed that ectively , supportno acids . The ORFno acids . The dom CL0015) . The RPR CL0015) . Indeed, CL0015) . The ali insects ; and so  the MFS . Further the MFS   and the brown marmorated stink bug . In addition, all the glucose transporters form a separate monophyletic group and the TRET from Escherichia coli (WP_077788896.1) forms the out group.As part of the characterization of Rhopr-TRET, we evaluated the spatial transcript expression of this transporter using various tissues from unfed adult females (baseline condition) using RT-qPCR. In accordance with the premise that the fat body is the main tissue involved in nutrient and energy metabolism, the highest expression of the s tissue . The Rhos tissue . The ress tissue , suggestTPS, m-Tre and s-Tre as well as of Rhopr-TRET in the fat body and ovaries. Except for TPS, the transcript expression pattern in both tissues is quite similar throughout the different time points of the unfed and fed condition. In the fat body, TPS transcript levels are lower during the unfed condition but rise significantly post-blood meal when vitellogenesis begins, with maximum seen 1 day after feeding  by day 1 after blood feeding and remain high over the 5 days analyzed , porcine insulin (P_In), and R. prolixus ILP1 (Rhopr-ILP1), and an insulin receptor kinase activator, bpV (phen). RT-qPCR reveals that after 3 h of incubation, only the treatment with bpV (phen) is able to induce a significant increase in Rhopr-TRET expression     or Rhopr-AKH  and insulin (using P_In) signaling in unfed females by injection of these hormones into females at 10 days post-ecdysis, when Rhopr-TRET expression in the ovaries is low  . Howevern = 3–4) . Rhopr-An = 3–4) , suggests is low . At 3 h  ovaries . As withal times .Rhopr-TRET transcript expression, we knocked down the receptors Rhopr-IR1 and Rhopr-AKHR, using RNAi in fed or unfed females . To confirm the potential regulation of Rhopr-TRET by hormones, we down-regulated Rhopr-IR1 and Rhopr-AKHR transcripts in unfed females and 1 day after dsRNA treatment, we injected porcine insulin (P_In) or Rhopr-AKH, as indicated (Rhopr-TRET expression in ovaries after P_In (dsRhopr-IR1/P_In) or Rhopr-AKH (dsRhopr-AKHR/Rhopr-AKH) stimulation to the same levels as those of control insects [dsARG/P_In and dsARG/Rhopr-AKH  at 3 h post-injection, but not in the ovaries   and 1 day after dsRNA treatment, we injected trehalose  to the same levels as those of control insects (dsARG/T)  . To confrehalose . InsectsdsARG/T) . This fiRhopr-AKHR transcript in the fat body and ovaries increases as the unfed condition advances, with higher levels at 30 days post-ecdysis. Accordingly, the Rhopr-AKH transcript expression in CNS also increases at 30 days post-ecdysis (Rhopr-AKHR significantly decreases with respect to controls (dsARG)   2 days pR. prolixus is of some importance to support successful embryogenesis  for energy generation. Although in some insects, such as the beet armyworm Spodoptera exigua, starvation induces an increase in circulating trehalose titers , coincident with the increase in trehalose in the ovaries, indicating that this carbohydrate could be transported from the hemolymph via Rhopr-TRET into the oocytes. These findings are not contradictory with those reported by TRET was first described in the anhydrobiotic insect, tebrates . Rhopr-Tsorption . Another hindgut . In R. panalysis . This upbyx mori . InteresRhopr-IR1 through RNA-mediated interference, and stimulation with exogenous insulins, show that insulin signaling is positively influencing Rhopr-TRET transcript expression in the ovaries of fed insects, a state where anabolic processes are required for egg formation and where it is known that the insulin signaling cascade is activated  transcriptional factor , Rhopr-AKH (3 h) appears to act faster than insulins (12 h), suggesting that the ovaries are more responsive to AKH than to insulin in an unfed state. Also, we evaluated the expression of the TPS transcript after manipulations to down- or up- regulate AKH and insulin signaling and the results show no significant differences compared to control insects. The same effect was observed in the beet armyworm S. exigua (arvation . In starugh TRET . Similarignaling . These fignaling . In a col factor . Recentll factor . Consideng cells . Further. exigua .R. prolixus (Rhopr-TRET transcript expression that could then result in mediating a TRET facilitated bidirectional transfer of trehalose: the release of trehalose from the fat body and the uptake by the ovaries, satisfying the physiological needs required for egg formation. The potential up-regulation of Rhopr-TRET transcript via Rhopr-AKH signaling during vitellogenesis is also suggested, as well as a positive feedback of hemolymph trehalose levels on insulin signaling. In the unfed condition, Rhopr-AKH signaling might be involved in the release of trehalose from the ovaries via Rhopr-TRET, thereby providing a source of energy for the insect during stressful situations such as starvation (R. prolixus, trehalose homeostasis and its hormonal regulation by insulin and AKH signaling could play critical roles in adapting to different nutritional conditions. Further experiments using biochemical or functional assays could possibility reveal the entire regulatory mechanism of the trehalose metabolism in triatomines.Although the effects of insulin and AKH signaling on nutrient mobilization have been reported in prolixus , b, the arvation  althoughThe original contributions generated for this study are included in the article/JL, AL, and IO designed the experiments and mapped out the manuscript. JL and HE-M performed the experiments. JL wrote the original draft of the manuscript and prepared all the figures. AL and IO edited, reviewed, and contributed to the writing of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This paper aims to compare the effects of three treatment modalities for a submacular hemorrhage (SMH) secondary to exudative age-related macular degeneration (AMD). Seventy-seven patients with an SMH were divided into three groups: small-sized (optic disc diameter (ODD) ≥ 1 to < 4), medium-sized  and large-sized . Patients received anti-vascular endothelial growth factor (anti-VEGF) monotherapy, pneumatic displacement (PD) with anti-VEGF or a vitrectomy with a subretinal tissue plasminogen activator (tPA) and gas tamponade based on the surgeon’s discretion. The functional and anatomical outcomes were evaluated. Among the 77 eyes, 45 eyes had a small-sized, 21 eyes had a medium-sized and 11 eyes had a large-sized SMH. In the small-sized group, all treatment modalities showed a gradual best-corrected visual acuity (BCVA) improvement with high hemorrhagic regression or displacement rates (over 75%). In the medium-sized group, PD and surgery were associated with better BCVA with more displacement than anti-VEGF monotherapy . In the large-sized group, surgery showed a better visual improvement with a higher displacement rate than PD (86% vs. 25%). Our findings demonstrated that visual improvement can be expected through appropriate treatment strategy regardless of the SMH size. In cases with a larger SMH, invasive techniques including PD or surgery were more advantageous than anti-VEGF monotherapy. Submacular hemorrhage (SMH) is one of the severe complications in patients with neovascular age-related macular degeneration (AMD). As it physically separates the neurosensory retina from the retinal pigment epithelium (RPE) layer, the resulting barrier effect interferes with the diffusion of nutrients and metabolites . Additio3F8) or sulfahexafluoride (SF6) can be used to push away the SMH from the fovea. A tissue plasminogen activator (tPA), a serine protease, dissolves the blood clot by cleaving the fibrin [Various treatment modalities for SMH have been developed. Expansile gas injection including perfluoropropane CF8 or sule fibrin . A surgie fibrin . Single A widely used treatment option for the SMH is anti-VEGF monotherapy. It has shown favorable results but severe cases were often excluded from previous studies ,7,8,9. THence, we sought to compare the anatomical and functional outcomes of practically selected treatment modalities depending on the hemorrhage size in patients with AMD complicated by an SMH. We also evaluated the possible prognostic factors for visual outcomes.A retrospective chart review of patients with an SMH from March 2015 to November 2018 at Yeungnam University Hospital was performed. The Institutional Review Board of the Yeungnam University Hospital approved the protocol. Data were collected from medical charts of patients who had an SMH involving the fovea secondary to exudative AMD. Additional inclusion criteria were age > 50 years and SMH thickness > 250 μm.The baseline evaluation included best-corrected visual acuity (BCVA) with a Snellen chart, dilated fundus examination, fundus photography and spectral domain optical coherence tomography (SD-OCT). An angiography with fluorescein or indocyanine green was performed to identify the underlying lesion.The fundus images were evaluated with ImageJ software  for Windows. The longest diameter of the SMH was measured and recorded as multiple values of the optic disc diameter (ODD). Patients were divided into three groups based on the size of the SMH: small-sized (≥ 1 to < 4-disc ODD), medium-sized  and large-sized  . The difUsing SD-OCT , a standard volume scan consisting of a 6 × 6 mm area was performed at the macular area and at points considered to have the largest SMH. A retinal specialist (Dong-Geun Park) reviewed all the images and measured the maximum height of the SMH using a manual ruler in the integrated software.3F8 gas with an intravitreal anti-VEGF injection simultaneously with or without an intravitreal tPA. When anti-VEGF therapy was combined with the tPA, aflibercept, which is known to be cleaved by the tPA [6 gas filling through a fluid–gas exchange.Patients were prescribed a treatment modality based on the surgeon’s judgement of optimal treatment. Twenty-nine patients were treated with an intravitreal anti-VEGF monotherapy . Twenty-five patients received pneumatic displacement (PD) using 0.3 cc of 100% C the tPA , was avo3F8 gas with an anti-VEGF injection (C3F8/anti-VEGF) was selected mainly for the medium-sized SMH and a PPV with a subretinal tPA and SF6 gas (PPV/tPA/ SF6) was preferred for the large-sized SMH, respectively. Additionally, parameters including location, thickness and duration of the hemorrhage were considered to achieve the best outcomes for each patient when choosing the treatment option. Every procedure was performed by a retinal surgeon (Min Sagong). Patients who received any gas injections were instructed to maintain a face-down position for 2–3 days.The surgeon applied these three strategies according to the SMH size. Anti-VEGF monotherapy was considered for the small-sized SMH preferentially. CIf a patient complained of decreased vision during the follow-up period, we performed thorough examinations to detect any sign of recurrence including a newly developed SMH, a small retinal hemorrhage, increased retinal thickness on an OCT, leakage on an angiography or decreased vision alone. Based on the surgeon’s decision, additional anti-VEGF injections were administered.The main outcomes were the degree of displacement or regression of the SMH from the fovea at 1 month after treatment; BCVA at 1, 3, 6 and 12 months after the initial treatment; prognostic factors for visual acuity; frequency of additional anti-VEGF injections; incidence of a recurrent SMH or breakthrough vitreous hemorrhage and other complications.p-value < 0.05 were considered significant.Statistical analyses were performed using IBM SPSS ver. 20.0 for Windows . BCVA was measured using a Snellen chart and converted to the logarithm of the minimum angle of resolution (logMAR) units for statistical analyses. An analysis of variance or a Kruskal–Wallis test was performed to determine the differences in the baseline parameters among the subgroups. If there was a difference, a post-hoc analysis with a Bonferroni correction was performed to identify the groups that caused that significance. Categorical variables among the subgroups or among the treatment modalities were analyzed by χ2 test or Fisher’s exact test. To identify factors that correlated with BCVA at 3 months and 12 months, a Pearson correlation test and analysis of covariance (ANCOVA) were performed. Variables with a p = 0.164), sex (p = 0.755), duration of symptoms (p = 0.653), lens status (p = 0.119) and proportion of anticoagulant usage (p = 0.068). The mean size of the SMH as an ODD was 2.45 ± 0.90, 5.09 ± 1.27 and 7.55 ± 2.27 in the small-, medium- and large-sized subgroups (p < 0.001), respectively. The mean SMH thickness in each group increased according to the SMH size . Regarding the AMD lesions, the small-sized group showed a higher percentage of polypoidal choroidal vasculopathy (PCV) (66.7%) than CNV (33.3%). In contrast, the large-sized group consisted of more CNV (72.7%) than PCV (27.3%). There was a statistical difference in the distribution of classifications among the three groups (p = 0.028). The treatment modalities were differently distributed in each subgroup (p = 0.011). Anti-VEGF monotherapy was applied mainly in the small-sized group (51.1%) and also applied in the medium-sized (28.6%) groups. A combination of PD with anti-VEGF and/or an intravitreal tPA injection was applied in all subgroups . A PPV with a subretinal tPA and SF6 gas, although also applied in the small- (22.2%) and medium-sized (28.6%) groups, was mainly applied in the large-sized group (63.6%).Among the 77 eyes of 77 patients with hemorrhagic AMD, 45 eyes had a small-sized, 21 eyes had a medium-sized and 11 eyes had a large-sized SMH. The baseline characteristics of the affected eyes are provided in p = 0.200; 3F8/anti-VEGF and a PPV/tPA/SF6 showed 75% and 100% of complete displacement, respectively. In the medium-sized group, anti-VEGF monotherapy showed poor anatomical outcomes (33% of complete regression) while the other two treatment methods showed acceptable results . In the large-sized group, the PPV/tPA/SF6 contributed to the high percentage of complete displacement (86%) but C3F8/anti-VEGF did not (25%) (The distribution of the SMH displacement did not significantly differ among the subgroups (ot (25%) .p = 0.015). The post-hoc analysis revealed a significant effect of the large-sized group when compared with that of the small-sized group (p = 0.014). The overall mean logMAR BCVA improved from 1.30 ± 0.83 at baseline to 1.03 ± 0.95 at 3 months (p = 0.040), 1.00 ± 0.93 at 6 months (p = 0.044) and 0.76 ± 0.83 at 12 months (p = 0.008). Each subgroup showed an improvement of mean BCVA at each follow-up; the difference in the baseline mean BCVA among the subgroups disappeared at 6 months (p = 0.145) and 12 months (p = 0.139) (The baseline mean BCVA differed among the groups (= 0.139) .3F8/anti-VEGF was used in 36.4% and a PPV/tPA/SF6 was employed in 63.6% of patients. The mean BCVA improved only in the PPV/tPA/SF6 group and decreased in the C3F8/anti-VEGF group  and between the BCVA at 3 months and 12 months . The SMH thickness also showed a positive correlation with BCVA at 3 months  and 12 months  between the two groups.To identify the effect of treatment modality on visual acuity during the follow-up, ANCOVA with baseline visual acuity as a covariance was performed, which is a known prognostic factor. However, different treatment modalities did not influence the visual outcomes. We divided patients into two groups based on the SMH duration (duration ≤ 14 days or > 14 days) and performed ANCOVA with baseline visual acuity as a covariant. There was a significant difference in visual outcomes (p = 0.886). The mean number of injections were 3.27 ± 2.06, 2.60 ± 1.45 and 3.00 ± 1.85 during 12 months in each group (p = 0.526).For 12 months, additional anti-VEGF injections were administered to 66.7%, 71.4% and 72.7% of eyes in each group . There w6.Breakthrough vitreous hemorrhage occurred in three (6.7%), one (4.8%) and four (36.4%) eyes of each group. One case of a recurrent SMH, one case of an RPE rip and one case of a macular hole were observed in the medium-sized group. . The RPEIn this study, we compared three treatment modalities for AMD complicated by an SMH. We included an extended spectrum of hemorrhage sizes as well as a larger number of patients than previous studies. Moreover, we divided our cases according to the SMH size and analyzed the treatment outcomes of three different modalities to identify their effects in each subgroup with homogeneity in terms of size.The overall mean BCVA improved significantly from the baseline during the 12 month follow-up period. However, different outcomes were observed according to the SMH size and treatment modalities. In the small-sized group, anti-VEGF monotherapy was performed on the majority of patients (51.1%) and it significantly improved the visual acuity. The two other treatment modalities also increased the mean BCVA and the magnitude of improvement showed no difference among the treatment modalities. This result may be related to the anatomical outcome that a small-sized SMH was effectively regressed by anti-VEGF only (75% of complete regression) without gas or vitrectomy. Consistent with our results, previous studies reported the efficacy of anti-VEGF monotherapy with a minimally invasive technique ,18,19. THowever, anti-VEGF monotherapy was no longer effective in the medium-sized group while the other two modalities increased the mean BCVA. Moreover, anatomical outcomes were worse with monotherapy (33% of complete regression) than with other modalities . In a retrospective study comparing anti-VEGF monotherapy with a combination of anti-VEGF, tPA and gas , the com6. Limited studies have reported the efficacy of a vitrectomy and subretinal tPA for SMH [6 showed a better outcome than PD only (changes of mean BCVA were −0.90 vs. +0.10 logMAR units) [6 (86%) but not with C3F8/anti-VEGF (25%). This result suggests that for a massive SMH, a vitrectomy with a subretinal tPA is more efficient than PD, showing better functional and anatomical outcomes.In this study, the only effective treatment option for visual improvement in the large-sized SMH was the PPV/tPA/SF for SMH . FassbenR units) . TreumerR units) . The meaThe amount of the SMH that has been considered a prognostic factor for visual outcomes  is cliniIn this study, a strong correlation of BCVA at 3 months with the BCVA at 12 months was identified, suggesting that optimal initial treatment and subsequent anti-VEGF retreatment may be helpful to maintain the initial visual gain. Treumer et al.  reported6 was a more invasive technique, intraoperative and other postoperative complications were rarely observed. One case of an RPE rip (4.3%), one of a macular hole (4.3%) and none of retinal detachment (RD) were observed, similar to that in previous studies  [6. All procedures were done by an experienced surgeon, which minimized complications. It was also helpful to try to find a suitable location for the subretinal injection location while avoiding large blood vessels and pigment epithelial detachment using multimodal images including fundus photography and OCT. The rarity of complications associated with the PPV/tPA/SF6 and its efficacy in the large-sized hemorrhage group could justify our treatment selection.Of the complications during the follow-up, vitreous hemorrhage was more common in the large-sized group (36.4%) than in other groups . However, the percentage of vitreous hemorrhage was not different among the treatment modalities . Although the PPV/tPA/SF% of RD) ,28,29. TOur study has several limitations. Due to the retrospective characteristics, the treatment modalities were not consistently applied in each group according to the SMH size, resulting selection bias. However, all judgments were made by one experienced surgeon to minimize bias in the choice of treatment options. In addition, due to the nature of the disease, the number of patients with a large SMT was small, which limited the statistical analysis. The scarcity of the disease and the fact that inadequate treatment in time can lead to severe vision loss make it difficult to conduct large-scale prospective studies for a limited period along with ethical issues. Therefore, in the study of rare diseases, the real-world data including many patients can be more important.In conclusion, the individualized treatment options for a SMH depending on the hemorrhage size showed acceptable anatomical outcomes with visual improvement in each subgroup. For the small-sized SMH, anti-VEGF monotherapy was useful. However, in the medium-sized SMH, anti-VEGF only was not effective and the rapid removal of the hemorrhage was required for a thick SMH. For a large-sized SMH, a vitrectomy with a subretinal tPA and expansile gas were the only effective treatment options. Our strategy of selecting the treatment modality according to the hemorrhage size could be a reference for the treatment of SMHs.


N,N’-dimethylacrylamide) (PDMA) as the hydrophilic block using bulk reversible addition-fragmentation chain transfer (RAFT) polymerization of o-nitrobenzyl acrylate using a PDMA macro-RAFT agent. The developed amphiphilic block copolymers have a suitable hydrophobic/hydrophilic ratio and can self-assemble into photoresponsive polymersomes for co-loading hydrophobic and hydrophilic cargos into hydrophobic membranes and aqueous compartments of the polymersomes. The polymersomes demonstrate a clear photo-responsive characteristic. Exposure to light irradiation at 365 nm can trigger a photocleavage reaction of o-nitrobenzyl groups, which results in dissociation of the polymersomes with simultaneous co-release of hydrophilic and hydrophobic cargoes on demand. Therefore, these polymersomes have great potential as a smart drug delivery nanocarrier for controllable loading and releasing of hydrophilic and hydrophobic drug molecules. Moreover, taking advantage of the conditional releasing of hydrophilic and hydrophobic drugs, the drug delivery system has potential use in medical applications such as cancer therapy.Due to a strong retardation effect of o-nitrobenzyl ester on polymerization, it is still a great challenge to prepare amphiphilic block copolymers for polymersomes with a o-nitrobenzyl ester-based hydrophobic block. Herein, we present one such solution to prepare amphiphilic block copolymers with pure poly (o-nitrobenzyl acrylate) (PNBA) as the hydrophobic block and poly ( Photo-responsive polymersomes  have attracted great interest as controlled drug delivery systems since they can realize spatial and temporal control of the release of the encapsulated therapeutics on demand via light irradiation ,2,3. TheAmong these photosensitive moieties, o-nitrobenzyl and its derivatives have gained intensive attention for the applications of controllable drug delivery ,24,25. oN,N’-dimethylacrylamide) (PDMA) as the hydrophilic block by bulk RAFT polymerization of o-nitrobenzyl acrylate using a PDMA macro-RAFT agent (PDMA–CTA). Shown in Herein, we present one solution to prepare an amphiphilic block copolymer with pure poly (o-nitrobenzyl acrylate) (PNBA) as the hydrophobic block and poly  PDMA30-b-PNBA97 is determined in 30-b-PNBA97 that can self-assemble into polymersomes is shown in 30-b-PNBA97 have a low molecular dispersity index of 1.25 with an average number molecular weight of 20.5 kg/mol.To fabricate a photosensitive polymer, a photoresponsive monomer NBA was synthesized according to the literature . Then, t30-b-PNBA97 was first monitored by UV-vis spectroscopy. Shown in 30-b-PNBA97 would transfer into a double hydrophilic block copolymer PDMA-b-PAA, as shown in To ensure that the developed polymersomes possess the photo-triggered disassembly for controlled drug release, the photo-responsive property of the PDMA30-b-PNBA97 copolymers in water/chloroform emulsions followed with complete removal of chloroform via gradual evaporation. The size of the polymersomes also was measured by dynamic light scattering (DLS) using a Zetasizer Nano-ZS from Malvern Instruments equipped with an He-Ne laser at a wavelength of 633 nm at 25 °C and the measurement of sucrose at a 173° detection angle. The results indicated the diameter of the polymersomes was about 200 ± 30 nm  and Nile red dye (NR), were co-loaded into the polymersomes and the photo-triggered co-release of these two types of drugs was evaluated. 2-Nitrobenzyl alcohol and acryloyl chloride was purchased from Shanghai Haiqu Chemical Co., Ltd. Nile red  and 2, 2′-azobisisobutyronitrile (AIBN) was purchased from Aldrich . Dichloromethane (DCM), chloroform, ethyl ether and tetrahydrofuran (THF) were purchased from Aladdin . Water was deionized with a Milli-Q SP reagent water system ) for a specific resistivity of 18.4 MΩ.cm. 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid (DMAP) was prepared according to a reported procedure . All the1H-NMR spectra were obtained using Deuterated Chloroform as a solvent and Tetramethylsilane as an internal norm on a Bruker 300 MHz spectrometer . The spectra were employed to determine the polymerization unit of the block copolymers. Measurements of Gel permeation chromatography (GPC) were carried out on a Waters device fitted with a photodiode array detector (PDA 996) and a refractive index detector (RI 410) . GPC setup was comprised of PL gel 10 mm MIXED-B columns using a single refractive index (RI) detector  and a series of narrow polystyrene as standards for the calibration curve. The eluent was THF, and the flow rate was 1.0 mL·min−1 at 40 °C. THF was used as the eluent at an elution rate of 1 mL/min, while polystyrene standards were used for calibration. Photoreaction of the block copolymer as a function of UV irradiation time was recorded on a Varian 50 Bio UV-vis spectrophotometer . The fluorescence emission spectra were recorded on a fluorescence spectrophotometer (Varian Cary Eclipse) . The morphologies of the polymer vesicles were inspected using a Hitachi H-7500 transmittance electron microscope (TEM)  operating at 60 kV. Regarding TEM measurement, a drop of the solution was spread on a TEM copper grid. The solution remained for 5 min and the excess solution was blotted off using filter paper and allowed to dry at room temperature overnight. 4. The product was collected by filtration and then purified by flash column chromatography on silica gel with DCM as an eluent, which was obtained as a light-yellow oil (1.3 g).Synthesis of 2-Nitrobenzyl Acrylate (NBA) Monomer. The monomer was synthesized according to a reported procedure with some modifications . Typical1H-NMR spectrum. The sample was obtained as a PDMA30 macroRAFT agent (PDMA-CTA).RAFT Synthesis of PDMA macro-RAFT agent. RAFT polymerization of DMA was conducted at 70 ℃, employing AIBN as the radical initiator and DMAP as the RAFT chain transfer agent. A typical example is as follows: DMA , DMAP , AIBN  were added to a 25 mL one-pot round flask. Then, 3 mL of anhydrous anisole was included to dissolve the blend. After purging 30 min with argon of high purity, the whole system was immersed into a preheated oil bath for 4 h at 78 ℃ with constant stirring. The crude product was purified by precipitating twice into excess ethyl ether, followed by vacuum-drying. The monomer conversion was approximately 100% as measured from the -b-PNBA Diblock Copolymer. PDMA-CTA was used as the chain transfer agent to prepare the block copolymer. PDMA-CTA , AIBN  were dissolved in NBA . After purging Argon for 30 min, the reaction was continued for 24 h at 80 ℃ with constant stirring. The product was purified by precipitating three times into excess cold hexane, followed by vacuum-drying overnight. The sample was obtained as a yellowish powder . GPC PDMA30-b-PNBA97.RAFT Synthesis of PDMAVesicle Preparation and Drug Loading/Photo-Controlled Release. Regarding self-assembly of the block copolymer in water, water was added to a solution of the polymer (5 mg per mL) in chloroform and the two-phase mixture was stirred vigorously for three days. After evaporation of the organic solvent, the aqueous phase was dialyzed against deionized water for 24 h. The final concentration of the block copolymer was 2.5 mg per mL.Doxorubicin Encapsulation. An aqueous solution of Doxorubicin hydrochloride (DOX) was added to a solution of the block copolymer (5 mg per mL) in chloroform and the two-phase mixture was stirred vigorously for three days. After evaporation of the organic solvent, the aqueous phase was dialyzed against deionized water for 24 h. Non-entrapped DOX was removed by dialysis against water for 3 days. The final concentration of the polymer was 2.0 mg per mL.Nile red Encapsulation. Twenty-four microliters of a 0.5 mg per mL stock solution of Nile red in dichloromethane was added to an empty vial, which was then placed under vacuum for at least two hours to ensure complete solvent removal. To this vial was added 1 mL of 2.5 mg per mL aqueous vesicular solution. This solution was stirred vigorously at room temperature for at least 4 h, and then filtered with a column of cotton.Drug loading efficiency (DLE) was calculated according to the following Equation (1):The DLE of DOX and Nile red were determined to be 81% and 75%, respectively.Photo-Controlled Release of a Hydrophilic Drug upon UV Irradiation. The drug release experiments were carried out as follows: A UV cell filled with 3 mL pure water was carefully closed with the dialysis cap whose membrane was immersed in water. Then, 0.3 mL of DOX-loaded vesicle solution, after UV irradiation or no irradiation, was poured into the dialysis cap. Sampling the solution underneath the dialysis membrane enabled the measurement of the fluorescence emission of DOX that is released from the vesicle solution and diffuses into the cell through the membrane. The release kinetics of DOX was recorded following irradiation for certain time periods.Photocontrolled Release of a Hydrophobic Drug upon UV Irradiation. The stock NR-loaded vesicular solution was a diluted system, and the fluorescence measurements were taken. The above procedure was repeated.30-b-PNBA97, with a suitable hydrophobic/hydrophilic ratio for self-assembly of polymersomes. The resultant polymersomes realized controlled dissociation upon light irradiation and were used to load and release hydrophobic and hydrophilic drugs at the same time in a controlled means by tuning light irradiation time. We expect that the reported strategy can greatly simplify the synthesis of other o-nitrobenzyl-based block copolymers for developing photoresponsive polymersomes based on o-nitrobenzyl as a smart drug delivery nanocarrier for controllable co-release of hydrophilic and hydrophobic drug molecules. To summarize, we developed a simple and general strategy to prepare amphiphilic block copolymers using o-nitrobenzyl acrylate as the starting material via a routinely-adopted controlled polymerization method. This method achieved a maximum conversion of 60 mol% of NBA in our polymerization conditions, endowing it with the ability to prepare an amphiphilic block copolymer, PDMA


The aim of this study was to investigate the association of visit-to-visit variability of hemoglobin A1c (HbA1c) and glycated albumin (GA) with the risk of lower extremity atherosclerotic disease (LEAD).We performed a prospective cohort study of 436 patients with type 2 diabetes (258 men and 178 women) with at least 3 measurements of HbA1c and GA prior to baseline investigation from the Department of Endocrinology and Metabolism, Shanghai Sixth People’s Hospital. Different HbA1c and GA variability markers were calculated. Multivariable Cox proportional hazard regression models were used to demonstrate the association between visit-to-visit HbA1c and GA variability and the risk of incident or progressive LEAD.P for trend = 0.042), respectively. When we used GA-VIM and GA-ARV values as exposures, similar positive associations with the risk of LEAD primary were found. Multivariate-adjusted HRs of LEAD for each 1 unit increase in GA-CV, GA-VIM and GA-ARV were 1.03 (95% CI 1.01–1.06), 1.32 (95% CI 1.03–1.69), and 1.07 (95%CI 1.01–1.15), respectively. However, there was no significant association between visit-to-visit variability of HbA1c and the risk of LEAD.During a mean follow-up period of 3.77 years, 112 participants developed LEAD. Multivariate-adjusted hazard ratios (HRs) of LEAD across tertiles of GA-CV values were 1.00, 1.06 , and 1.71 (95% CI 1.07–2.73) (Visit-to-visit variability of GA may be an optimal biomarker in relation to LEAD risk among patients with type 2 diabetes. Lower extremity atherosclerotic disease (LEAD) is one of the life-threatening complications of diabetes. LEAD and the subsequent vascular occlusion, gangrene and lower extremity amputation seriously affect the quality of life of patients and increase the economic burden , 2. HoweAmong patients with diabetes, long-term exposure to hyperglycemia is an important risk factor for the development of macrovascular complications. Glycated hemoglobin (HbA1c) has been widely used as a marker for glycemic control , 4. PrevGiven the above reason, we speculate that visit-to-visit variability of GA can provide more information on long-term glycemic control than HbA1c. The association between visit-to-visit variability of GA and LEAD in patients with type 2 diabetes remains uncertain. The present study aimed to investigate the association between visit-to-visit variability of GA and the risk of LEAD in patients with type 2 diabetes.This is a prospective and dynamic cohort study of patients with type 2 diabetes aged 18–80 years who were admitted to the Department of Endocrinology and Metabolism, Shanghai Sixth People’s Hospital from January 2011 to September 2019. All patients were previously confirmed with type 2 diabetes by the 1999 World Health Organization criteria [All participants underwent routine physical examination to measure height, body weight, waist circumference and blood pressure. Body mass index (BMI) was calculated as body weight in kilograms divided by square of height in meters. Waist circumference was measured by a tape around the horizontal plane between the inferior costal margin and the iliac crest on the mid-axillary line with the subject in a standing position. Blood pressure was measured twice with a mercury sphygmomanometer, and the average value of the two measurements was taken. History of comorbidities and prescriptions was collected including anti-hypertensive medications, glucose-lowering medications, lipid-lowering medications and antiplatelet medications. Using data collected on the patient’s self-reported smoking status, we classified the patients into 2 groups: current smokers, non-current smokers including ever smokers and never smokers. Current alcohol consumption was defined as weekly alcohol consumption over 140 g in men and 70 g in women . Mean vaAll subjects were fasted overnight for 8–10 h before venous blood sampling. Serum uric acid (UA), serum creatinine (Scr), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), fasting blood glucose (FPG) and fasting C peptide (FCP) were measured using the same methods that have been previously described .β. β stands for a standardized coefficient of fitted power function with SD and mean. ARV was calculated as the average value of the difference between consecutive measurements [The updated mean value, standard deviation (SD), variability independent of the mean (VIM), and the average real variability (ARV) of GA and HbA1c were calculated for each participant within 2 years before baseline date collection. CV of GA and HbA1c was calculated as SD/mean × 100%. VIM was calculated as SD/meanurements . In thisThe primary outcome (LEAD) was the composite outcome of either incident LEAD or progressive LEAD confirmed by Doppler ultrasonography. The femoral artery, deep femoral artery, popliteal artery, superficial femoral artery, anterior tibial artery, posterior tibial artery and peroneal artery were measured bilaterally. Arterial intima-media thickness (IMT) was measured as the distance between the intima-lumen and the media-adventitia . AtherosP < 0.05. All statistical analyses were performed using IBM SPSS Statistics for Windows, version 24.0 .The associations between visit-to-visit variability of GA and HbA1c  and the risk of the primary outcome were analyzed by use of Cox proportional hazards models. CV, VIM and ARV of GA and CV of HbA1c were evaluated in the following 2 ways: as tertiles and as a continuous variable (per 1-unit increase). These visit-to-visit GA and HbA1c variability metrics were included in the models as dummy variables, and the significance of the trend across categories of GA and HbA1c was tested in the same models by giving an ordinal numeric value for each dummy variable. The proportional hazards assumption in the Cox model was assessed with graphical methods and with models including time-by-covariate interactions. In general, all proportionality assumptions were appropriate. All analyses were conducted after adjusting for age and sex, and then a backward stepwise selection procedure was used for candidate covariates in the multivariable model including BMI, waist circumference, diabetes duration, systolic blood pressure (SBP), lipid profile , eGFR, smoking status, current alcohol drinking, insulin therapy, anti-hypertensive therapy, aspirin, statins therapy, and mean value of HbA1c. Finally, we included diabetes duration, smoking status, eGFR, HDL-C, mean value of HbA1c and use of aspirin in the multivariable-adjusted model. Statistical significance was considered to be P = 0.324), and fewer men . The baseline characteristics are listed in Table A total of 539 patients were enrolled in the study at baseline with a loss of 103 during follow-ups. Finally, 436 patients with type 2 diabetes were included into the final analysis. Compared with patients with diabetes excluded in the present study, the patients with type 2 diabetes included in the present study had the similar age , respectively  HRs of the primary outcome across tertiles of GA-CV values were 1.00, 1.06 , and 1.71 (95% CI 1.07–2.73) , the linear association between GA-CV, GA-VIM and incident LEAD remained significant with multivariable adjustments (HR 1.04 (1.01–1.07) for CV and 1.44 (1.05–1.97) for VIM). Similar results were observed for the outcome of progressive LEAD with additionally GA-ARV significantly associated with the outcome as well.For the analysis of the secondary outcome  during the comprehensive management of diabetic complications.GA itself was reported to be closely related to cardiovascular disease and its subtypes in patients with diabetes  and evenCompared with HbA1c, GA is not affected by hemoglobin, and can more accurately reflect the glycemic control in cases of anemia, hemoglobinopathy, dialysis , and preWe calculated several metrics in the present study including CV, VIM and ARV of GA and HbA1c based on at least 3 measurements within 2 years prior to baseline as indicators for evaluating long-term glycemic fluctuations. Notably, SD, CV, adj-SD, VIM, ARV and variability scores have all been used in previous studies , 23, 24.The mechanism of an increased risk of macrovascular complications caused by increased long-term blood glucose fluctuations is not yet clear. Studies have potentially supported the hypothesis of oxidative stress , endotheThe strength of this study included the originality, the prospective study design and the robust analysis. Limited studies have investigated the association between visit-to-visit variability of GA and the risk of LEAD in patients with type 2 diabetes. There are some limitations that need to be pointed out. First, this was a single center study with a relatively small sample size and few outcome cases. Second, our analyses adjusted for some confounding factors, but unmeasured factors such as other related chronic diseases, dietary factors, and physical activity could not be evaluated. Moreover, the index date of the primary outcome was determined by the date of lower extremity vascular ultrasonography. It is thus not yet possible to accurately obtain the time of lesion progression. However, the median follow-up time interval  in this study was 1.14  years, which is in line with the screening frequency recommended in guidelines for patients under a high risk of LEAD . The subIn conclusion, visit-to-visit variability of GA predicts LEAD risk among patient with type 2 diabetes, independent of HbA1c levels. In contrast, visit-to-visit variability of HbA1c was not an independent factor for LEAD among patients with type 2 diabetes. Visit-to-visit variability of GA may be closely related to LEAD among patients with type 2 diabetes.Additional file 1: Table S1. Hazard ratios for the primary outcome based on different HbA1c variability metrics. Table S2. Hazard ratios for the secondary outcome based on different GA variability metrics. Figure S1. Flow chart of the study population.


Background: Many patients with type 2 diabetes mellitus (T2DM) do not have adequate glycemic control, leading to poor patient outcomes and high healthcare costs.Objective: This prospective pragmatic clinical trial evaluated V-Go, a wearable insulin delivery device, compared with standard treatment optimization (STO) among insulin-treated patients with T2DM in a realworld, community-based practice setting.Methods: Study sites, rather than individual patients, were randomized to V-Go or STO via cluster randomization. Patients were treated according to routine clinical practice and followed up to 4 months. T2DM medications and supplies were purchased utilizing usual insurance and co-pay systems. The primary analysis was an unadjusted treatment group comparison of glycosylated hemoglobinA1c (HbA1c) change from baseline to end of study (EOS). A cost of therapy analysis was completed on patients who had received comparable baseline T2DM treatment with multiple daily basal-bolus insulin injections (MDI).Results: Analysis included 415 patients  enrolled from 52 US sites. Mean baseline HbA1c (9.6%) was higher in V-Go  than STO . HbA1c decreased from baseline to EOS in both V-Go  and STO ; V-Go had significantly larger decrease (p=.002). V-Go had a significant reduction (p<.001) in mean insulin total daily dose , not seen in STO (0.72 U/kg baseline and EOS). The MDI group included 95 (56.2%) V-Go and 113 STO (45.9%) patients. Mean baseline HbA1c was significantly higher in V-Go (9.9%) than STO (9.4%). V-Go also experienced larger decrease in HbA1c from baseline (-1.0%) than STO (-0.36%) (p=.006) with a decrease in TDD, while STO TDD remained unchanged. EOS mean per patient per day cost of diabetes treatment was lower for V-Go ($30.59) vs STO ($32.20) (p=.006). V-Go was more cost effective than STO .Conclusions: This pragmatic clinical trial demonstrated improved HbA1c levels, lower cost, and decreased insulin dose in patients with T2DM initiating V-Go vs STO in a real-world community-based practice setting. Observed baseline HbAlc indicated use of V-Go in more difficult to manage diabetes patients. Type 2 diabetes mellitus (T2DM), affecting approximately 30 million Americans,Patient self-reported adherence to insulin regimens ranges from as low as 43% up to 86%, with more flexible regimens and lower patient costs associated with improved adherence.Poorly controlled T2DM is associated with higher costs, and even small improvements in glycemic control could result in substantial cost savings for third party payers.The V-Go device has demonstrated efficacy in controlled clinical settings,The current study was designed to fill this gap in evidence. It sought to prospectively assess the effectiveness of the V-Go insulin delivery device compared with standard insulin delivery in adults with T2DM in a real-world, community-based practice setting.SM). The HIRD is a large, clinically rich, administrative healthcare database with a broad population base representative of the US population overall.a priori did not want to use V-Go could not participate in the study.This was a pragmatic clinical trial conducted in adults with T2DM treated with insulin in a real-world, community-based practice setting between September 2013 and April 2016. Physician practice study sites were recruited from clinicians identified as providing care for adult insulin-treated T2DM patients through administrative claims analysis of the HealthCore Integrated Research Database  HbA1c 8% to 14%; be currently using insulin therapy with total daily dose of 30 U to 120 U; be willing to go to the physician’s office for follow-up visits and to complete the patient-reported outcome instrument; be willing and able to understand and sign a written informed consent form. Patients were excluded from the study if they had current or planned use of an insulin pump for diabetes management; they were currently participating in any clinical study; they were pregnant, lactating, or intending to become pregnant; were currently using chronic systemic steroids; or they had a diagnosis of type 1 diabetes.a)-Diabetes Device, a brief treatment satisfaction questionnaire, at baseline and EOS.Consistent with the pragmatic design, there were very few study-specific evaluations. Upon enrollment, study physicians treated patients according to routine clinical practice and followed them for a minimum of 3 months, and up to 4 months, to collect study data. Demographic, clinical , and insulin regimen data were collected at baseline. Any on-study changes to insulin therapy were recorded as patients presented to the physician as part of standard care. Patients had an end of study (EOS) visit approximately 3 months + 30 days after baseline to collect weight, HbA1c, and insulin regimen data. The EOS had a wide window of up to 30 days after the 3-month time point so as to not impose a schedule on study physicians’ routine clinical practice. Patients completed the Treatment-Related Impact Measures  prior to implementation of any study procedures.Demographic and clinical data were collected either directly from patients or abstracted from the patients’ medical records and entered into the electronic Case Report Form (eCRF) at study visits (baseline and EOS) by trained study personnel. Patients completed the TRIM-Diabetes Device questionnaire by pen and paper at baseline and EOS, and site study personnel entered the completed forms into the eCRF. Insulin regimen data were collected at baseline and any time there was a change to a patient’s insulin regimen throughout the study.The primary endpoint was change in HbA1c from baseline to EOS. Baseline HbA1c was defined as the most recent diagnostic HbA1c laboratory value within 4 weeks prior to the baseline visit. EOS HbA1c was defined as that obtained closest to EOS, at least 3 months (+30 days) after the baseline visit. Due to the pragmatic design, in some cases HbA1c tests were performed outside the baseline or EOS visit windows. For these cases, the HbAlc obtained closest to the respective time point was used. The secondary endpoints included patient treatment satisfaction  and Device Bother (3 times). Each item utilizes a 5-point Likert scale and assesses patient satisfaction with their device over the past two weeks. Responses are summed and transformed into a 0-100 scale, where higher scores represent higher device satisfaction.TDD  was calculated in U per day and U per kg body weight for baseline, EOS, and change from baseline. Baseline TDD was calculated from the insulin regimen prior to commencement of any study procedures, ie, prior to initiation of V-Go for patients randomized to the V-Go arm. EOS TDD was the insulin regimen collected closest to EOS. For patients whose insulin regimen did not change during the study, the baseline regimen was used for EOS TDD.The cost endpoint was the mean per patient per day (PPPD) cost of diabetes treatment, including all concomitant diabetes medications and devices, using Wholesale Acquisition Cost (WAC) at EOS. Cost ($) of diabetes treatment per 1% drop in HbA1c was calculated based on the sample means: WAC costs for all diabetes treatments divided by mean change in HbA1c.All analyses were performed using SAS® version 9.4 or higher computer software. The significance level used in all statistical analyses was 5% (two-sided). While physician practices were the unit of randomization for this study, patients were the unit of analysis. Descriptive statistics  were used to summarize patient characteristics, treatments, and outcomes for the study population overall and by treatment group. All change from baseline variables were calculated as absolute change = EOS – baseline. Between-group differences in outcome variables were evaluated using independent t-tests. Within-group differences  were evaluated using paired t-tests.a priori. As a result of these restrictions, all data analysis was unadjusted; observations of recorded testing  were not controlled, but rather reported as collected.Statistical analyses were consistent with pragmatic research and used the observations of patient treatments unobstructed by adjustments in the methodology and statistical approach, so as not to interfere with the real-world setting of both treatment patterns practiced by clinicians, as well as the analysis methods determined to be appropriate The analysis population was defined as patients who met all inclusion criteria, none of the exclusion criteria, and who had an EOS HbA1c measurement.Additional analyses were completed on the population with MDIs, a subset of the analysis population that had comparable baseline diabetes treatment with 3 or more daily insulin injections including both basal and bolus insulin injections.A total of 585 patients with T2DM  were identified from 52 clinical sites . Of these, 415 patients (70.9%) had a valid post-baseline EOS HbA1c value and were included in the analysis population . Reasons for not completing the study included declining study participation/refusing V-Go initiation (45 V-Go), early study withdrawal or loss to follow-up , V-Go related discontinuations (32 V-Go), and death (1 STO). The median follow up time was 97 days for the analysis population overall, and did not differ between V-Go  24 days) and STO  treatment groups (p=.692).2, range 17.9 kg/m2 to 102.7 kg/m2). Baseline HbA1c levels ranged from 7.9% to 14.2% with an overall group mean of 9.6%. The mean TDD at baseline was 71.9 U/day (or 0.74 U/kg) and was similar between treatment groups. Nearly all patients had at least one comorbid condition (96.4%) with hypertension (83.1%) and hyperlipidemia (74.5%) being the most commonly reported.The majority of patients included in the analysis were white (62.4%) and one quarter of the population was black/African American (25.8%). More women (54%) than men comprised the study population, with a mean age of 59.8 years . There was a wide variation in baseline weight  and body mass index  compared with the STO group .Although the baseline mean TDD insulin was similar between the treatment groups, the average TDD in the V-Go group decreased significantly from baseline to EOS , while the TDD for the STO group remained unchanged (Table 2).A significant decrease in mean HbA1c from baseline (9.6%) to EOS (8.9%) was observed in the overall study population . Patients in the V-Go group experienced a significantly larger decrease than those in the STO group .Concomitant diabetes medication use was evaluated for both treatment groups. A higher proportion of patients in the STO group reported using more concomitant diabetes medications in addition to insulin compared with the V-Go group at EOS. Although a high proportion of patients in both groups used 0 to 2 concomitant diabetes medications at EOS (95.2% V-Go and 89.0% STO), a smaller percentage of patients in the V-Go group used 3 or 4 concomitant diabetes medications than those in the STO group (4.8% V-GO and 10.9% STO).Physicians reported that 93.5% of patients in the V-Go group used the device as directed. Overall, patients rated both the V-Go and standard diabetes delivery devices highly, with mean scores in the 70s and 80s on a 100-point scale, where higher scores indicate higher device satisfaction. Baseline mean TRIM-Diabetes Device scores, which reflect the device used in the 2 weeks prior to study enrollment, were statistically significantly lower in the V-Go group than in the STO group, with between-group mean differences ranging from 5.2  to 11.6  points in total and subscale scores. However, on average, the mean TRIM-Diabetes Device scores increased from baseline to EOS for the V-Go group, indicating improvement in patient-reported outcomes, but did not appreciably change for the STO group. (Table 3). The largest difference was observed in the Bother subscale, where the V-Go group averaged a 4.8-point increase from baseline (p=.035 for within-V-Go group difference) compared with a 0.4-point decrease for the STO group (p=.043 for between-group difference). The V-Go group also had a statistically significant increase, or improvement,  in the Total TRIM-Diabetes Device score whereas the STO group had a mean decrease of 0.2 in the score. (Table 3).Cost and effectiveness analyses were completed on patients  who had comparable baseline diabetes treatment with 3 or more injections per day including both basal and bolus insulin injections. In this MDI group, comprising 50% of the overall study group, patients in the V-Go group had a higher baseline HbA1c (9.9%) than the STO group (9.4%). The V-Go group experienced a significantly larger decrease in HbA1c from baseline (-1.0%) than the STO group . This decrease was observed with a reduction in mean TDD administered to patients in the V-Go group from baseline (75 U/day) to EOS (55 U/day). In contrast, the mean TDD of the STO group remained unchanged from baseline to EOS (77 U/day). Additionally, the mean PPPD cost of diabetes treatment , calculated using WAC, was lower for the V-Go ($30.59) compared with the STO ($32.20) group. Improved cost effectiveness was seen in V-Go-treated patients, with WAC costs of $24.02 per 1% drop in HbA1c, compared with $58.86 for patients in the STO group (Table 4).This prospective, pragmatic clinical trial provides real-world evidence of increased glycemic control and patient treatment satisfaction in adults with T2DM initiating treatment with the V-Go insulin delivery device, compared with patients using standard delivery of insulin and treatment optimization. Additionally, patients in the V-Go group had decreased insulin requirements and costs compared with the standard therapy group. This is the first pragmatic trial to specifically study the impact of the V-Go insulin delivery device compared to standard treatment delivery.On average, the V-Go and STO groups both demonstrated improved glycemic control as evidenced by significant decreases in HbA1c levels in the study population overall as well as by treatment group within the 4-month treatment period. A larger decrease in HbA1c was observed in the V-Go group than in the STO group, suggesting additional benefit beyond routine follow-up. However, the mean baseline HbA1c levels were higher in the V-Go group than in the STO group, suggesting greater diabetes disease severity in patients in the V-Go group, which may have contributed to observed differences in study outcomes between treatment groups. Previous research suggests the greatest gains in glycemic control upon switching to V-Go are seen in patients with higher prior HbA1c levels.Overall, the study population had a mean baseline HbA1c level of 9.6%. While high, it is also indicative of the real-world nature of this pragmatic clinical study and highlights the unmet need for improved therapy for this population of adults with T2DM. The elevated HbA1c levels are also consistent with previous retrospective studies targeting sub-optimally controlled diabetes, in which mean baseline HbA1c levels ranged from 8.9% to 9.6%.Although patients in the V-Go group experienced significant decreases in their insulin requirements, these were accompanied by significant reductions in HbA1c values from baseline to EOS. Patients in the V-Go group reported a 24% reduction in TDD U/day and a 25% reduction in TDD U/kg while patients in the STO group reported no changes in TDD. Our observation of decreased insulin utilization after initiation of V-Go is consistent with previous researchPatients in the V-Go as well as the STO group were generally satisfied with their insulin delivery device at baseline and EOS, with average TRIM-Diabetes Device scores in the 70s and 80s on a 100-point scale. Patient satisfaction scores at baseline, ie, satisfaction associated with their insulin delivery device used prior study enrollment, was on average lower in V-Go group compared with STO. However, on average, patient satisfaction scores in the V-Go group increased from baseline to EOS while scores in the STO group remained unchanged. The largest improvement in V-Go scores was in the Bother subscale (mean change of 4.8 points), which represented both a significant change from baseline and a significant difference from the change in the same subscale in the STO group (mean change -0.4 points from baseline to EOS). This finding is consistent with prior research indicating positive patient perceptions of V-Go,The observation of improved glycemic control, coupled with decreased insulin requirements in patients initiating treatment with V-Go compared with STO, persisted in an analysis of the MDI subset. The MDI subset had comparable baseline diabetes treatment, however, as in the overall cohort, mean baseline HbA1c levels were higher in the V-Go group than in the STO group and may have contributed to the observed differences in study outcomes between treatment groups. Similar to the analysis of the overall study population, both V-Go and STO groups in the MDI subset experienced decreases in HbA1c from baseline to EOS. The decrease in the V-Go group was significantly larger than in the STO group  and was similar to the overall V-Go population (-1.0%). In the MDI subset, the mean TDD of the V-Go group decreased significantly from baseline to EOS while it remained unchanged in the STO group. Additionally, the calculated cost of diabetes treatment per 1% improvement in HbA1c was 59% lower in V-Go compared with STO in the MDI subset.a priori data analysis plan is methodologically appropriate.While this pragmatic clinical trial had several strengths, including increased generalizability of the results across ethnicities and races due to the size and composition of the study population, limitations should be noted. There were observed differences in baseline characteristics between treatment groups. However, because these differences were reflective of the pragmatic nature of this study, no statistical controls were put into place to adjust or artificially equate these samples. Some may argue that data analysis adjusted for baseline variation, rather than unadjusted data, would be conducted and reported. However, given the pragmatic design and the real-world context, the unadjusted data analysis following the In particular, differences indicated greater diabetes disease severity in patients in the V-Go treatment group, possibly due to a bias of more advanced diabetes and perhaps less adherent patients initiated on V-Go, given the cluster randomization scheme. Therefore, the contribution of diabetes disease severity to observed treatment group differences must be considered. Additionally, a higher percentage of patients in the V-Go group did not complete the study compared to patients in the STO group, which may have in part been due to greater flexibility in STO treatment or personal disinclination towards a wearable device. The study objective was to compare insulin delivery via V-Go versus standard of care, as determined by the treating physician according to routine clinical practice. As such, patients enrolled in the STO group were allowed to add or change therapy during the study as consistent with their clinician’s standard practice, whereas patients initiating V-Go must have initiated and maintained V-Go for the study duration. This design constraint may have introduced a differential adherence allowance between treatment groups, which could have contributed to the observed difference in study outcomes.The pragmatic design had few study-specific evaluations, but simply being followed for study participation may have influenced treatment outcomes. However, study-specific evaluations (other than V-Go-specific instructions) were the same between treatment groups. While both treatment groups had significant improvements in overall HbA1c levels, the decrease in HbA1c levels in the V-Go group was significantly greater than that in the STO group, indicating the device was likely a factor in the result. The follow-up period was relatively short (up to 4 months) and maximal effectiveness of the intervention was likely not attained. The significantly higher baseline HbA1c in the V-Go group compared with the STO group plus previous research suggest the greatest gains in glycemic control after V-Go initiation are observed in patients with higher initial HbA1c levels.This is the first large-scale pragmatic study to evaluate the V-Go device in a real-world community-based setting. The population was largely similar to the general diabetes population receiving insulin, with high baseline HbA1c levels and other comorbidities as demonstrated in large epidemiological studies.Funding for this study was provided by Valeritas, Inc. Mark Cziraky, Thomas Wasser, and Elizabeth Apgar are employees of HealthCore, Inc., which received funding from Valeritas, Inc. for the conduct of the study. Lynn Nowak was an employee of HealthCore, Inc. at the time of the study. Scott Abbott and Matt Nguyen are employees of Valeritas, Inc. Study design, data collection and interpretation, manuscript drafting and review, and decision to publish were performed solely by the authors.© Novo Nordisk, August 2008; TRIM-D Device contact information and permission to use: MAPI Research Trust, Lyon, France.TRIM-D DeviceE-mail: PROinformation@mapi-trust.org – Internet: www.mapi-trust.org


The membrane surface morphology was characterized by SEM, and the compositions were magnified using a high-resolution transmission electron microscope (HRTEM). Notably, the membranes showed excellent substance stability in an alkali medium and in grafting tests performed in a QM-soluble solvent. The ED experiment indicated that the as-prepared membrane exhibited better HSO3− separation performance than the state-of-the-art commercial Neosepta AMX  membrane.Ion-exchange membranes are the core elements for an electrodialysis (ED) separation process. Phase inversion is an effective method, particularly for commercial membrane production. It introduces two different mechanisms, i.e., thermal induced phase separation (TIPS) and diffusion induced phase separation (DIPS). In this study, anion exchange membranes (AEMs) were prepared by grafting a quaternized moiety  through brominated poly  (BPPO) via the TIPS method. Those membranes were applied for selective bisulfite (HSO Open-ended distinct characters of membranes have been considered and adopted in industrial uses. Ion-exchange membranes (IEMs) have significantly influenced the purification and separation process via electrodialysis (ED)  and otheSince brominated poly  (BPPO) has access to benzyl halide groups, an alternative is possible regarding chloromethylation for subsequent quaternization. Moreover, BPPO polymer could be formed from another monomer through grafting without any catalysts . Due to Besides, naphthalene is a fused aromatic compound and has conjugated π-electrons. Among all the derivatives, 1-naphthol has a much higher melting point and comparatively low molar mass . In prevIn this innovation, a fresh derivative QM compound was used as functionalized using a 1-naphthol aromatic alcohol. Then, the moiety was quaternized over iodomethane. After that, BPPO was homogeneously dissolved in n-methyl-2-pyrrolidone (NMP), the solution was heated at 60 °C for about 4 h, and QM solution in NMP was added at 12 h, allowing grafting to occur through the quaternized membrane formed via the TIPS. Those membranes exhibiting grafting through anion exchange membranes (AEMs) were confirmed through the DIPS test in different solvents and with a high-resolution transmission electron microscope (HRTEM). Furthermore, uniquely developed membranes were favorable for bisulfite anion separation through ED.1H NMR spectrum analysis (Bruker 400 MHz) as illustrated in 2 peak at 4.20 to 4.40 showed a broad peak for the moiety, and a little sharp peak was observed for the QM independently. Herein, the QM had formed a methylene group that had an influence on the proton spectrum [The sound synthesis of the QM was carried out by following the procedure illustrated in spectrum . Moreove−1, which is ascribed to the C-N stretching vibration for the QA groups [−1, but it was absent in the BPPO. Meanwhile, an absorption peak at around 2950 cm−1 was facilitated by -CH3 functional groups. A broad absorption peak was detected at around 3450 cm−1 in the FTIR-ATR results, which was missing for BPPO. This defines the stretching vibration of 0-H bands due to the presence of water in the AEMs [The quaternized ammonium (QA) groups inside the membrane core developed during grafting were confirmed by the FTIR-ATR and results displayed in A groups . This bathe AEMs .SEM was used to view the assembly of tiny microscopic phenomena. The surface images were distinguished from each other see . FurtherConsequently, the prepared quaternized membranes were homogeneous in nature. For ED, a dense morphology of the MM is required, which can be observed from the microscopic images. Furthermore, to enhance the selectivity between hydrogen ions, hydroxide ions, and other anions in an acid–base interaction through the MM, the non-interconnected nanopores, and amphoteric topology could be considered. HRTEM was utilized to investigate the structural morphology of prepared BPPO-g-QM (1:0.60) quaternized AEMs. The images from the HRTEM are shown in 2O absorption and thickness properties.The ED process acts as effector for electro-potential. Annexing outcomes show that the ion exchange capacity (IECs) of prepared quaternized AEMs increase with an increase in QMs in the MM. Enriching the concentration of QMs in the MM was found to decrease them. In short, the hydrophilicity of the membrane might be improved. Moreover, the density of QMs in the MM affects the H2O absorption, hydrous angle, and chemical stability are essential properties (available in The transport number (TN) was found to be increased from the BPPO-g-QM (1:0.30) to the BPPO-g-QM (1:0.60) membrane by increasing the consistency of the QMs in the MM, except that BPPO-g-QM (1:0.86) showed a lower TN then BPPO-g-QM (1:0.60). Hence the diffusion of co-ions across the membrane is suppressed by rising quaternary ammonium groups (QAGs). The BPPO-g-QM (1:0.60) membrane achieved the highest TN (0.81) among all. This could be attributed to the highest content of QAGs through the optimum suppression of co-ion diffusion. Besides, the BPPO-g-QM (1:0.86) membrane obtained a lower TN due to the degree of substitution in the MM. The IEC, H3 solution in dilute cell (DC) and concentration cell (CC) were changed in both cases, as shown in An ED test was performed at a laboratory scale to test the bisulfite anion separation performance of the BPPO-g-QM (1:0.60) membrane 3 groups. Such a characteristic might be correlated with the increasing ion-exchange content in the MM. However, the BPPO-g-QM (1:0.60) membrane was utilized in the ED system to separate HSO3− anions from aqueous solution, showing a better desalination rate and current efficiency than Neosepta AMX.Background chemistry would occur upon water dissociation (splitting) with AEMs that, through the weak primary group, resulted in reversible protonation and deprotonation . The fix therein . Water d3I), sodium hydrogen sulfite (NaHSO3), and trichloromethane (CHCl3) were purchased form Sinopharm Chemical Reagent Co. Ltd. (Shanghai China). Deionized water was used as necessary. Shandong Tianwei Membrane Co. Ltd. (Weifang China) provided the BPPO polymer. A 52% aryl degree of substitution was determined by 1H NMR (see 1-naphthol (99%) was bought from Shanghai Aladdin Industrial Co. Ltd. (Shanghai China). Formaldehyde solution (37–40%), DMSO, NMP, hexane, ethyl acetate, ethanol absolute, methanol, iodomethane  was added gently to the corresponding 1-naphthol and dimethylamine  at room temperature with continued stirring for 12 h. The product was extracted with CHCl3, dehydrated with anhydrous sodium sulfate, and concentrated in an exhausted drying oven. Finally, a hexane/AcOEt eluent (1:6) silica gel was used for the customized purification of the crude product by column chromatography. The QM was prepared through a single process shown in previous work [3 at room temperature for 48 h. After completion of the reaction, the precipitate was washed with ether (absolute) a minimum of five times and concentrated in a desiccator. Thereafter, the quaternary amine formation was verified by 1H NMR, as shown in A standard method was implemented to prepare the moiety as reported . Formaldous work . The moiFor the polymeric membrane, the TIPS and DIPS techniques were used. The membrane morphology, nascent porosity, and surface formation are affected by the thermodynamics of the polymer solution . TIPS isThe refined BPPO polymer was dissolved in NMP (10 wt.%) at ~ 5 °C to produce a solution. It was transferred onto the top of a horizontal glass plate, which was decorated with the metal plate with uniform heat through increasing the temperature up to 60 °C for ~ 4 h. Then, QM  equivalent was added in 1 mL of NMP solvent before mixing gently to ensure analogy . TherefoDIPS modifies a polymer solution by three methods: immersion precipitation, vapor adsorption, and solvent evaporation . Herein,2 gas flow, with a heating rate of 10 °C/min. The mechanical properties were determined with an Instron Universal tester (DMA Q800) . The chemical stability was calculated by the deviation of the IEC and weight value before and after immersion in a 2 M NaOH solution at room temperature for a particular duration. The inner morphology was imaged by SEM . Furthermore, the distribution of the elements on the surface SEM-(EDS) was studied. Additionally, AFM (Dimension Icon SPM) was used to observe the phase resolution and roughness. The thermic steadiness of the membrane was inspected with a TGA50H as regarding the temperature (25–800 °C) under continued NAn innovative AEM was successfully prepared with BPPO and QMs, applying the TIPS technique by grafting through copolymerization. The DIPS test was used to test the conduction of a QM-soluble solvent and showed excellent grafting. SEM images predicted the compact structures. Most importantly, HRTEM images showed the grafting configuration. The AEM seems to be reliable in terms of thermal and alkaline persistence. For separation of the ions, the prepared membrane was applied for bisulfite anion separation instated of AMX (Neosepta) and exhibited a better desalination rate. The principal bisulfite anion desalination rate (3.2%) obtained with the BPPO-g-QM (1:0.60) membrane was higher than that obtained with the commercial AMX (1.4%). The current efficiency (37.36%) achieved with the BPPO-g-QM (1:0.60) membrane was better than that with the commercial AMX (16.20%). However, the 1-naphthol base QM is a new functional material. So far, extending grafting through the 1-naphthol base with QMs in the MM has been demonstrated by HRTEM (showing a 2D clear conformation of QMs) construction, ultimately showing excellent performance of bisulfite anion separation in ED. Moreover, anion separation is the most complicated job due to different aspects. Consequently, particular anions, grafting structures, and strengthening of conjugated π-electrons through grafting should be analyzed in detail in the ion-separation field in our future work.